The liberation of arachidonic acid from cell membrane phospholipids is the first step along the biosynthetic pathway leading to the eicosanoids (prostaglandins, leukotrienes and others), which are potent mediators of inflammation and other physiological responses. It is generally accepted that glucocorticoid steroids exert their anti-inflammatory properties by blocking the cell's ability to liberate free arachidonic acid from membranes. Over the past decade several lines of evidence have appeared to indicate that the 85-kDa group IVA cytosolic phospholipase A2 (cPLA2-alpha) is critical for arachidonic acid release in agonist-stimulated pro-inflammatory cells including neutrophils and macrophages. cPLA2-a action in mammalian cells is highly regulated. Regulation elements known to date are: 1) the phosphorylation of the enzyme; 2) the change in cytosolic calcium concentration; 3) the composition of the membrane bilayer to which cPLA2-alpha binds. A consensus of how these factors contribute to cPLA2-a regulation has not yet been reached. A major goal of our proposed studies is to carry out a detailed analysis of the binding of cPLA2-alpha to membranes in vitro and to determine the enzymatic activity under different experimental conditions. We will then extend these studies to the intracellular situation by monitoring cPLA2-alpha membrane binding and enzymatic activity in living mammalian cells. Our lab has recently shown that another phospholipase A2, the secreted enzyme, can augment the action of cPLA2-alpha in mammalian cells. The molecular basis for this crosstalk between the two phospholipases A2 will be investigated. In the past few years, a number of cPLA2-a paralogs have been spotted in the human and mouse genomes. The functions of these paralogs remain to be established. We will use a structure-guided approach to redesign a potent inhibitor of cPLA2-alpha in an effort to obtain potent inhibitors of the cPLA2-alpha paralogs. Such compounds will be useful as a tool to establish the functions of these paralogs in mammalian cells.